This invention relates generally to a medium for the in vitro culture of animal cells. More specifically, the invention is a defined nutrient medium capable of supporting serum-free culture. Further, serum-derived proteins typically present in defined (i.e., serum free) media may be replaced with non-protein-based cell growth enhancers and a protein supplement which is non-serum-derived. This unique medium has been designed specifically for supporting animal cell growth without supplementation with serum or serum-derived proteins Excellent cell growth is achieved and the medium is
of a wide variety of cell lines and cell types.
For in vitro culture, a medium must, of course, supply all essential nutrients for the cells: vitamins, amino acids, lipids, nucleic acid precursors, carbohydrates, trace elements, and bulk ions. Historically, basal nutrient media were designed to support cell growth only after being supplemented with a biological extract, e.g., serum or embryo extracts. Serum, in particular, proved to be an effective supplement, presumably because it contains the necessary growth- and multiplication-promoting factors in physiologically acceptable concentrations. Examples of basal nutrient media of this type are Eagle's basal medium (BME), the composition of which is recited in U.S. Pat. No. 3,450,598 (Welsh et al.), and Dulbecco's Modified Eagle's (DME) medium, the composition of which is recited in Table II of Ham et al., "Media and Growth Requirements," Methods of Enzymology, (1978). DME medium, which contains relatively high concentrations of the essential amino acids and sugars, is representative of the commercially available media formulated for the mass culture of cells with serum supplementation.
With growing sophistication in cell culture techniques, factors present in serum or other biological extracts have been identified. It is now possible to grow mammalian cells in a serum-free environment, by supplementing a basal nutrient medium with defined proteins necessary for cell growth and multiplication. Ham's F12 medium, the composition of which is given in Table II of Ham et al., supra, contains low concentrations of the essential amino acids and sugars, and includes lipids, nucleic acid derivatives, vitamins and nonessential amino acids.
It is now generally accepted that a readily obtainable and sufficiently complex basal nutrient medium for mass culture of cells in low serum concentrations can be fabricated by mixing DME and F12 media. Such mixtures, when supplemented with the appropriate protein factors, can also support the serum-free growth of many cell types. Barnes et al., "Methods for Growth of Cultured Cells in Serum-Free Medium," Analytical Biochem., Vol. 102, pp. 255-70 (1980), describes examples of both approaches.
Several commercially available nutrient media are based on mixtures of DME, F12 and/or other media such as those listed in Table II of Ham et al., supra. However, simple mixtures of existing commercial media are by no means optimal for culturing all cell lines and medium preparations therefore have been targeted largely to particular cell lines or cell types. Wolfe et al., "Continuous Culture of Rat C6 Glioma in Serum-Free Medium," J. Cell Biol , Vol. 87, pp. 434-41 (1980), teaches the use of a 3:1 DME-to-F12 mixture, supplemented with trace elements, and further supplemented with the following defined proteins insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and serum-spreading factor (vitronectin). Similarly, a serum-free basal nutrient medium is disclosed in U.S. Ser. No. 029,577, "Basal Nutrient Medium for Cell Culture," (Wolfe), filed Mar. 24, 1987, which is supplemented with defined proteins such as albumin, iron-saturated transferrin, insulin, vitronectin and fibroblast growth factor.
With the increasing use of cultured mammalian cells to produce biologicals (e.g., monoclonal antibodies and genetically engineered proteins), there is an increasing demand for chemically defined, serum-free media. Purification of the desired cellular product is greatly complicated by the presence of serum or serum-proteins. It is therefore desired to reduce the protein content of the culture medium to a few defined compounds from which the monoclonal antibody or other cellular product can be separated more readily. It is also desired to reduce the protein content of the media as much as possible.